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Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

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Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). (E) Bar graph representing weakening of cell–cell adhesion (measured as cell monolayer fragmentation) for Pkp2, 3, and double KD. Error bars are ± SEM. ***p < 0.001 (ANOVA, Bonferroni). Note: Pkp2-3 double KD caused excessive fragmentation, so only up to 800 fragments were counted.
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Figure 1: Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). (E) Bar graph representing weakening of cell–cell adhesion (measured as cell monolayer fragmentation) for Pkp2, 3, and double KD. Error bars are ± SEM. ***p < 0.001 (ANOVA, Bonferroni). Note: Pkp2-3 double KD caused excessive fragmentation, so only up to 800 fragments were counted.

Mentions: To determine the role of Pkp3 in desmosome structure and function, we introduced Pkp3 and, for comparison, Pkp2 small interfering RNA (siRNA) pools (for analysis of individual sequences of the Pkp3 pool, see Supplemental Figure S1, A, B, and D–F) into SCC9 cells. This resulted in nearly total loss of respective targets without affecting the steady-state expression of other desmosome components, including compensatory expression of other plakophilins (Figure 1A). In Pkp3 knockdown (KD), localization of other desmosome components at cell–cell interfaces was aberrant. Desmoglein 2 (Dsg2), desmocollin 2 (Dsc2), and Pkp2 all exhibited a more discontinuous punctate distribution at cell–cell interfaces, as observed by immunofluorescence analysis of confluent monolayers (Figure 1B). In addition, Pg showed similar, albeit less pronounced disruption (Figure 1B). Similarly, ultrastructural comparison of Pkp3 KD cells with control cells revealed the presence of tightly clustered desmosomes (Figure 1C) separated by long stretches of membrane with no desmosomes. Moreover, Pkp3 KD cells exhibited significant variability in desmosome length and width compared with control cells, with some very large desmosomes present (Figure 1, C and D). Changes in desmosome morphology and a reduction in desmosome number correlate with weaker cell–cell adhesion (McMillan and Shimizu, 2001), so we next tested whether the strength of cell–cell adhesion was affected in Pkp2- and Pkp3-deficient cells. Cell monolayers lifted as a single sheet by using the enzyme dispase were subjected to sheer stress. Ablation of either protein increased the cell sheet fragmentation by approximately eightfold, with double knockdown resulting in complete loss of monolayer integrity (Figure 1E).


Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). (E) Bar graph representing weakening of cell–cell adhesion (measured as cell monolayer fragmentation) for Pkp2, 3, and double KD. Error bars are ± SEM. ***p < 0.001 (ANOVA, Bonferroni). Note: Pkp2-3 double KD caused excessive fragmentation, so only up to 800 fragments were counted.
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Related In: Results  -  Collection

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Figure 1: Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. ***p < 0.001 (t test). (E) Bar graph representing weakening of cell–cell adhesion (measured as cell monolayer fragmentation) for Pkp2, 3, and double KD. Error bars are ± SEM. ***p < 0.001 (ANOVA, Bonferroni). Note: Pkp2-3 double KD caused excessive fragmentation, so only up to 800 fragments were counted.
Mentions: To determine the role of Pkp3 in desmosome structure and function, we introduced Pkp3 and, for comparison, Pkp2 small interfering RNA (siRNA) pools (for analysis of individual sequences of the Pkp3 pool, see Supplemental Figure S1, A, B, and D–F) into SCC9 cells. This resulted in nearly total loss of respective targets without affecting the steady-state expression of other desmosome components, including compensatory expression of other plakophilins (Figure 1A). In Pkp3 knockdown (KD), localization of other desmosome components at cell–cell interfaces was aberrant. Desmoglein 2 (Dsg2), desmocollin 2 (Dsc2), and Pkp2 all exhibited a more discontinuous punctate distribution at cell–cell interfaces, as observed by immunofluorescence analysis of confluent monolayers (Figure 1B). In addition, Pg showed similar, albeit less pronounced disruption (Figure 1B). Similarly, ultrastructural comparison of Pkp3 KD cells with control cells revealed the presence of tightly clustered desmosomes (Figure 1C) separated by long stretches of membrane with no desmosomes. Moreover, Pkp3 KD cells exhibited significant variability in desmosome length and width compared with control cells, with some very large desmosomes present (Figure 1, C and D). Changes in desmosome morphology and a reduction in desmosome number correlate with weaker cell–cell adhesion (McMillan and Shimizu, 2001), so we next tested whether the strength of cell–cell adhesion was affected in Pkp2- and Pkp3-deficient cells. Cell monolayers lifted as a single sheet by using the enzyme dispase were subjected to sheer stress. Ablation of either protein increased the cell sheet fragmentation by approximately eightfold, with double knockdown resulting in complete loss of monolayer integrity (Figure 1E).

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

Show MeSH
Related in: MedlinePlus